UCP2 is highly expressed in pancreatic alpha-cells and influences secretion and survival

Abstract

In pancreatic beta-cells, uncoupling protein 2 (UCP2) influences mitochondrial oxidative phosphorylation and insulin secretion. Here, we show that alpha-cells express significantly higher levels of UCP2 than do beta-cells. Greater mitochondrial UCP2-related uncoupling was observed in alpha-cells compared with beta-cells and was accompanied by a lower oxidative phosphorylation efficiency (ATP/O). Conversely, reducing UCP2 activity in alpha-cells was associated with higher mitochondrial membrane potential generated by glucose oxidation and with increased ATP synthesis, indicating more efficient metabolic coupling. In vitro, the suppression of UCP2 activity led to reduced glucagon secretion in response to low glucose; however, in vivo, fasting glucagon levels were normal in UCP2(-/-) mice. In addition to its effects on secretion, UCP2 played a cytoprotective role in islets, with UCP2(-/-) alpha-cells being more sensitive to specific death stimuli. In summary, we demonstrate a direct role for UCP2 in maintaining alpha-cell function at the level of glucose metabolism, glucagon secretion, and cytoprotection.

Fig. 1.

Expression of UCP2 in dispersed pancreatic islets. Dispersed islets from UCP2^+/+ (A and B) and UCP2^−/− (C and D) mice were immunostained for UCP2 (red), and either glucagon (A and C, green) or insulin (B and D, green). Light (Left) and merged images (Right) are shown in the lower rows.

Fig. 2.

Expression of UCP2 in pancreatic cell lines. (Ai) MIN6 cells were immunostained for UCP2 (red) and insulin (green). (Aii) α-TC6 cells were stained for UCP2 (red) and glucagon (green). (Aiii) α-TC6 cells were stained for UCP2 (green) and mitochondria (red). Light (Left) and merged (Right) images are shown in the lower rows. (B) Western blot analysis of cytosolic, membrane, and mitochondrial fractions of MIN6 and α-TC6 cells with equal protein loading. Q, fractions prepared by using Qproteome Cell Compartment Kit; IM, fractions prepared by using Mitochondria Extraction Kit. (C) RT-PCR analysis of UCP1, 2, and 3 mRNA levels in MIN6 and α-TC6 cells (n = 6).

Fig. 3.

Glucose-stimulated alterations in the mitochondrial membrane potential in islets and pancreatic cell lines. (A and B) Islets dispersed from UCP2^+/+ and UCP2^−/− mice were cultured for 1–3 days. The ΔΨ_m-dependent Rh123 fluorescent signal (in relative fluorescence units, RFU) in selected regions of interest was obtained after the addition of 20 mM glucose and maximal mitochondrial polarization. The ΔΨ_m was then dissipated by the addition of the respiratory inhibitor NaN₃ (1 mM). (A) Representative kinetic traces of the fluorescent signal in small (non-β) cells are shown. (B) Quantitation of these traces is shown (n = 21–54 cells per condition; the experiment was repeated in islets taken from three animals per group on different days). (C) A representative trace showing the effects of the UCP2 activators OA and HNE on ΔΨ_m as determined by safranin fluorescence. (D) Quantitation of these traces and the effect of the UCP2 inactivator GDP. Values are mean ± SEM (n = 5; ∼30 cells measured per cell type in each experiment). *, P < 0.05; **, P < 0.01.

Fig. 4.

Effect of UCP2 knockdown on mitochondrial uncoupling in α-TC6 cells. (A) UCP2 protein levels were evaluated by costaining cells with specific antibodies against UCP2 (red) and glucagon (green). Merged images are shown, and yellow represents colocalization. (B) UCP2 mRNA levels were evaluated by RT- PCR in UCP2- vs. control-siRNA cells (n = 3). (C) Effect of UCP2 modulators OA, HNE, and GDP on ΔΨ_m were evaluated (n = 5). *, P < 0.01. Representative kinetic traces are shown in Fig. S3B.

Fig. 5.

Effects of UCP2 on ATP synthesis, respiration, and cellular ATP levels in pancreatic islets and cell lines. (A) Effect of UCP2 modulators OA, HNE, and GDP on ATP synthesis (ATP/O) in α-TC6 transfected with control- or UCP2-siRNA (i) or α-TC6 compared with MIN6 cells (ii). Values are mean ± SEM (n = 5). (B) State IV (V₄) respiration rate was measured in control- or UCP2-siRNA-transfected α-TC6 cells. Values are mean ± SEM (n = 5). (C) ATP levels were evaluated in islets (20 islets per sample) isolated from UCP2^+/+ and UCP2^−/− mice (n = 4–6 mice per group) (i) and α-TC6 cells transfected with control- and UCP2-siRNA (n = 5) (ii). Both islets and α-TC6 cells were pretreated with high glucose (HG, 25 mM) for 15 min followed by incubation with either HG or low glucose (LG, 0 mM) for 1 h. *, P < 0.05; **, P < 0.01.

Fig. 6.

Effect of UCP2 on glucagon secretion and pancreatic α-cell mass. Glucagon (A) and insulin (B) secretion from isolated islets in response to changes in glucose concentration (n = 8 for UCP2^−/− mice; n = 6 for UCP2^+/+ mice). Islets were preincubated in 25 mM glucose for 15 min and then switched to 25 mM glucose (HG) or 2.8 mM glucose (LG) for 1h. For secretion in the presence of arginine (10 mM), islets were incubated with HG (11 mM) or LG (1 mM) for 1h (n = 7 for UCP2^−/− mice; n = 6 for UCP2^+/+ mice). (C) Glucagon secretion was assessed in α-TC6 cells transfected with UCP2- or control-siRNA in the presence of 25 mM (HG) or 2.8 mM (LG) glucose (n = 3). (D and E) Fasting plasma glucagon (D) (n = 11 mice per group) and intracellular glucagon levels (E) (n = 6 mice per group) of isolated islets are shown. (F) α-cell mass was determined in pancreatic sections from UCP2^+/+ (n = 4) and UCP2^−/− (n = 5) mice. The glucagon-positive area was calculated and normalized to the islet area. *, P < 0.05; ***, P < 0.001.

Fig. 7.

Effect of UCP2 on islet cell viability under different treatments. (A) Islets from UCP2^−/− and UCP2^+/+ mice were dispersed (experiment n = 3–6; animal n = 3–8 per group; 623.6 ± 115.8 cells counted per treatment). Insulin negative (i) or positive (ii) cells were treated with medium (10% FBS and 11.5 mM glucose), serum-starved (LSHG) (0.01% FBS and 11.5 mM glucose), cytokines (a combination of IL-1, TNF-α, and IFN-γ at 100 ng of each per milliliter) in culture medium, or PA (0.5 mM) in medium. (B) α-TC6 cells transfected with UCP2- or control-siRNA were seeded at a high or low density and cultured in medium containing 0.05% FBS for three days. Cell viability was determined by using an XTT assay (n = 4). (C) Cell apoptosis was evaluated in α-TC6 cells transfected with UCP2- or control-siRNA by Western blot analysis for activated caspase-3 after treatment as indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.